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摘要：群体感应是细菌间联系的重要方式。基于luxR-AHL的群体感应转录调节组近年来得到了很多关注。前期研究表明：R蛋白对lux box的正向调控作用与营养条件有密切关系，在氨基酸饥饿情况下，R蛋白对lux后续启动子的转录甚至起了阻遏作用。本实验将不同类型的（严谨型和非严谨型）启动子与lux box组合起来，安装在pPOPGFPuv2质粒上，并且研究在氨基酸饥饿情况下荧光表达情况。质粒拼接过程进展顺利，两个片段都成功获取，并与三个不同的Linker进行最后的连接。19892
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关键词：群体感应；基因调控；启动子；荧光表达；lux box
The change of gene regulatory sequences lux box under different nutritional conditions
Abstract：Quorum-Sensing is an important way of contact between bacteria. Because quorum-sensing transcriptional regulators group of LuxR-AHL has got a lot of attention in recent years. Early studies show: R protein on the positive regulation of lux box and nutritional conditions are closely related, in the case of lack amino acid, R protein on transcription of lux subsequent promoter repressor even played a repressor role. In my experiment, I will link up promoter of different types (stringent and non-stringent type) to lux box, after that be linked to pPOPGFPuv2 plasmid, and study the condition of fluorescent expression under the lack amino acid. Plasmid splicing process is progressing smoothly, two fragments were successfully obtained, and compared with three different Linker would last connection.
Key Words：Quorum-Sensing；gene regulation；promoter；fluorescence expression；lux box
目 录
1 前言 1
1.1 细菌的群体感应 1
1.2 N-酰基高丝氨酸内酯 1
1.3 基因调控序列对lux box的影响 2
1.4 国内外的发展趋势 2
1.5 本研究主要内容及研究意义 3
2 材料与方法 4
2.1 材料与仪器设备 4
2.1.1 菌种 4
2.1.2 实验材料 4
2.1.3 主要仪器和设备 5
2.1.4 培养基及试剂的制备 6
2.2 实验方法 6
2.2.1 菌种的扩大培养 6
2.2.2 提取质粒 6
2.2.3 琼脂糖凝胶电泳 7
2.2.4 PCR扩增 8
2.2.5 质粒的酶切 9
2.2.6 琼脂糖凝胶回收 10
2.2.7 酶切产物的连接 11
3 实验结果与分析 12
3.1 提取质粒 12
3.2 验证提取的质粒为pPOPGFPuv2质粒 12
3.2.1 用MLUI酶酶切验证 12
3.2.2 PCR扩增验证 13
3.2.3 荧光反应验证 14
3.3 PCR扩增 15
3.4 PCR小片段939bp的酶切 16
3.5 PCR大片段4272bp的酶切 18
3.6 目的条带割胶回收 20
3.7 酶切产物的连接 20
4 结论 22
致谢 23
参考文献 24
1.前言